Genetic regulation of muscle fiber diversity. Abstract. The long-term objective of our research is to define the factors which regulate differentiation of the diverse skeletal muscle types which are found in animals. Distinct muscle fiber-types are specified in order to engender muscles with specialized characteristics, yet the molecular underpinnings of this specification process have yet to be defined. The Drosophila system has proven useful in defining mechanisms of muscle specification and muscle differentiation, and in addition the Drosophila adult thorax contains skeletal muscle fibers of two distinct types. We therefore propose to define the molecular bases of development for the adult Drosophila muscles, with the expectation that the processes that we uncover will contribute important new information into skeletal muscle developmental mechanisms, and will also define how distinct muscle fiber phenotypes arise. In the current period of funding, we have begun to define in detail how the regulatory factor Myocyte enhancer factor-2 (MEF2) controls muscle development in the Drosophila adult. While our data indicate an important requirement for MEF2 in adult myogenesis, we also found that a large number of adult muscle- specific structural genes are regulated independently of MEF2. In this application we plan to connect MEF2 function to adult muscle differentiation through two broad and complementary aims. In Aim 1, we shall identify the mechanism by which MEF2 functions in mediating adult muscle formation, including the factors which act alongside MEF2 in adult myogenesis. This will be achieved by determining the ability of MEF2 mutants to rescue adult and embryonic muscle development, by identifying regions of MEF2 that have tissue-specific functions, and by carrying out a genetic enhancer screen to identify loci whose haploinsufficiency exacerbates Mef2 hypomorphs. In Aim 2, we shall identify and characterize the regulatory factors responsible for the expression of adult muscle structural genes, via analyses of adult muscle-specific enhancers that we have identified, and by utilizing a new RNAi-based screen to identify genes which are required for the activity of adult muscle enhancers. The overall results of our experiments will provide a comprehensive view of how the complex muscles of the Drosophila adult are built. Given the strong evolutionary conservation in developmental regulatory processes between Drosophila and mammals, our studies will provide basic molecular mechanisms for how distinct skeletal muscle types differentiate and acquire fiber-specific properties. The developmental regulatory networks that we define in the Drosophila system will be an essential framework upon which to build our understanding of mammalian muscle development, disease, and repair. PUBLIC HEALTH RELEVANCE: Human muscles contain multiple fiber types, yet there is still much to learn in defining how these fiber types arise and how their fate is controlled. Moreover, a number of human muscle diseases preferentially affect one class of muscles over another. By defining mechanisms for how individual muscle arises in the body, our studies will provide a deeper understanding of how some muscles might be more sensitive to the development of pathologies, and how the symptoms of such diseases might be ameliorated.